Dorsal raphe serotonergic neurons preferentially reactivate dorsal dentate gyrus cell ensembles associated with positive experience.

Major depressive disorder (MDD) is among the most common mental illnesses. Serotonergic (5-HT) neurons are central to the pathophysiology and treatment of MDD. Repeatedly recalling positive episodes is effective for MDD. Stimulating 5-HT neurons of the dorsal raphe nucleus (DRN) or neuronal ensembles in the dorsal dentate gyrus (dDG) associated with positive memories reverses the stress-induced behavioral abnormalities. Despite this phenotypic similarity, their causal relationship is unclear. This study revealed that the DRN 5-HT neurons activate dDG neurons; surprisingly, this activation was specifically observed in positive memory ensembles rather than neutral or negative ensembles. Furthermore, we revealed that dopaminergic signaling induced by activation of DRN 5-HT neurons projecting to the ventral tegmental area mediates an increase in active coping behavior and positive dDG ensemble reactivation. Our study identifies a role of DRN 5-HT neurons as specific reactivators of positive memories and provides insights into how serotonin elicits antidepressive effects.

Median raphe serotonergic neurons projecting to the interpeduncular nucleus control preference and aversion.

Appropriate processing of reward and aversive information is essential for survival. Although a critical role of serotonergic neurons in the dorsal raphe nucleus (DRN) in reward processing has been shown, the lack of rewarding effects with selective serotonin reuptake inhibitors (SSRIs) implies the presence of a discrete serotonergic system playing an opposite role to the DRN in the processing of reward and aversive stimuli. Here, we demonstrated that serotonergic neurons in the median raphe nucleus (MRN) of mice process reward and aversive information in opposite directions to DRN serotonergic neurons. We further identified MRN serotonergic neurons, including those projecting to the interpeduncular nucleus (5-HTMRN→IPN), as a key mediator of reward and aversive stimuli. Moreover, 5-HT receptors, including 5-HT2A receptors in the interpeduncular nucleus, are involved in the aversive properties of MRN serotonergic neural activity. Our findings revealed an essential function of MRN serotonergic neurons, including 5-HTMRN→IPN, in the processing of reward and aversive stimuli.

Diff-ATAC-STARR-Seq: A Method for Genome-Wide Functional Screening of Enhancer Activity in Vivo.

Transcriptional regulatory elements, including promoters and enhancers, play a key role in the cell-type specific regulation of the transcriptome. Application of rapidly evolving genetic tools, such as optogenetic/chemogenetic actuators and fluorescent reporters to elucidate the function of cell subtypes in vivo necessitates cell-type specific promoters or enhancers. In this context, methods for genome-wide functional screening of cis-regulatory elements, including enhancers, are of utmost importance. In this study, we describe a novel method for genome-wide functional screening of enhancer activity in vivo with minimal handling. Application of the method to cells from different brain structures and subsequent differential analysis allow identification of active enhancers in the target tissue or brain structures. To demonstrate proof of concept, we applied this method to samples from the dorsal raphe nucleus (DRN) and the medial prefrontal cortex of the mouse brain and successfully identified six enhancers with highly biased activity towards the dorsal raphe nucleus. Considering that these two structures consist of largely similar cell types whereas serotonin and dopamine neurons exist only in the DRN, our results confirm the validity of this method in identifying cell-type specific and brain-structure specific enhancers. Overall, this method will be helpful in identifying cis-regulatory elements suitable for cell-type specific manipulations.

Autism Spectrum Disorder Model Mice Induced by Prenatal Exposure to Valproic Acid Exhibit Enhanced Empathy-Like Behavior via Oxytocinergic Signaling.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by core symptoms, including impairments in social behavior and repetitive interests. Recent studies have revealed that individuals with ASD also display decreased empathy, ultimately leading to difficulties in social relationships; however, another report indicated that individuals with ASD have enhanced emotional empathy. Nonetheless, the neurobiological mechanisms underlying altered empathy in individuals with ASD remain unclear. In this study, we assessed empathy-like behaviors in valproic acid (VPA)-treated mice-a mouse model of ASD with observational fear learning. We then investigated the brain regions and signaling systems responsible for the altered empathy-like behaviors in VPA-treated mice. As a result, mice prenatally exposed to VPA displayed increased empathy-like behaviors, which were not attributed to altered sensitivity to auditory stimuli or enhanced memory for pain-related contexts. Immunohistochemical analysis revealed that the number of c-Fos positive oxytocinergic neurons in the paraventricular nucleus of the hypothalamus (PVN) was significantly higher in VPA-treated mice after observational fear learning. Finally, we found that pretreatment with L-368899, an antagonist of the oxytocin receptor, repressed the empathetic behavior in VPA-treated mice. These results suggest that VPA-treated ASD model animals showed increased emotional empathy-like behaviors through the hyperactivation of PVN oxytocinergic neurons for the first time. Further investigation of this hyperactivity will help to identify extrinsic stimuli and the condition which are capable of activation of PVN oxytocinergic neurons and to identify novel approach to enhance oxytocin signaling, which ultimately pave the way to development of novel therapy for ASD.

Prediction of pharmacological activities from chemical structures with graph convolutional neural networks.

Many therapeutic drugs are compounds that can be represented by simple chemical structures, which contain important determinants of affinity at the site of action. Recently, graph convolutional neural network (GCN) models have exhibited excellent results in classifying the activity of such compounds. For models that make quantitative predictions of activity, more complex information has been utilized, such as the three-dimensional structures of compounds and the amino acid sequences of their respective target proteins. As another approach, we hypothesized that if sufficient experimental data were available and there were enough nodes in hidden layers, a simple compound representation would quantitatively predict activity with satisfactory accuracy. In this study, we report that GCN models constructed solely from the two-dimensional structural information of compounds demonstrated a high degree of activity predictability against 127 diverse targets from the ChEMBL database. Using the information entropy as a metric, we also show that the structural diversity had less effect on the prediction performance. Finally, we report that virtual screening using the constructed model identified a new serotonin transporter inhibitor with activity comparable to that of a marketed drug in vitro and exhibited antidepressant effects in behavioural studies.

The Role of Dorsal Raphe Serotonin Neurons in the Balance between Reward and Aversion.

BACKGROUND: Reward processing is fundamental for animals to survive and reproduce. Many studies have shown the importance of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons in this process, but the strongly correlative link between the activity of DRN 5-HT neurons and rewarding/aversive potency is under debate. Our primary objective was to reveal this link using two different strategies to transduce DRN 5-HT neurons. METHODS: For transduction of 5-HT neurons in wildtype mice, adeno-associated virus (AAV) bearing the mouse tryptophan hydroxylase 2 (TPH2) gene promoter was used. For transduction in Tph2-tTA transgenic mice, AAVs bearing the tTA-dependent TetO enhancer were used. To manipulate the activity of 5-HT neurons, optogenetic actuators (CheRiff, eArchT) were expressed by AAVs. For measurement of rewarding/aversive potency, we performed a nose-poke self-stimulation test and conditioned place preference (CPP) test. RESULTS: We found that stimulation of DRN 5-HT neurons and their projections to the ventral tegmental area (VTA) increased the number of nose-pokes in self-stimulation test and CPP scores in both targeting methods. Concomitantly, CPP scores were decreased by inhibition of DRN 5-HT neurons and their projections to VTA. CONCLUSION: Our findings indicate that the activity of DRN 5-HT neurons projecting to the VTA is a key modulator of balance between reward and aversion.

Identification of neuron-type specific promoters in monkey genome and their functional validation in mice.

Viral gene delivery is one of the most versatile techniques for elucidating the mechanisms underlying brain dysfunction, such as neuropsychiatric disorders. Due to the complexity of the brain, expression of genetic tools, such as channelrhodopsin and calcium sensors, often has to be restricted to a specified cell type within a circuit implicated in these disorders. Only a handful of promoters targeting neuronal subtypes are currently used for viral gene delivery. Here, we isolated conserved promoter regions of several subtype-specific genes from the macaque genome and investigated their functionality in the mouse brain when used within lentiviral vectors (LVVs). Immunohistochemical analysis revealed that transgene expression induced by the promoter sequences for somatostatin (SST), cholecystokinin (CCK), parvalbumin (PV), serotonin transporter (SERT), vesicular acetylcholine transporter (vAChT), substance P (SP) and proenkephalin (PENK) was largely colocalized with specific markers for the targeted neuronal populations. Moreover, by combining these results with in silico predictions of transcription factor binding to the isolated sequences, we identified transcription factors possibly underlying cell-type specificity. These findings lay a foundation for the expansion of the current toolbox of promoters suitable for elucidating these neuronal phenotypes.

TRPM2 confers susceptibility to social stress but is essential for behavioral flexibility.

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel and a member of the TRP channel superfamily that acts as a sensor of intracellular redox states. TRPM2 is widely distributed in many tissues and highly expressed in the brain, but the physiological roles of TRPM2 in the central nervous system remain unclear. In this study, TRPM2-deficient mice were examined in a series of behavioral tests. TRPM2-deficient mice did not significantly differ from wild-type littermates in muscle strength, light/dark transition test, rotarod, elevated plus maze, social interaction, prepulse inhibition, Y-maze, forced swim test, cued and contextual fear conditioning, and tail suspension test. In the Barnes circular maze, TRPM2-deficient mice learned the fixed escape box position at similar extent to wild-type littermates, suggesting normal reference memory. However, performance of the first reversal trial and probe test were significantly impaired in TRPM2-deficient mice. In the T-maze delayed alternation task, TRPM2 deficiency significantly reduced choice accuracy. These results indicate that TRPM2-deficient mice shows behavioral inflexibility. Meanwhile, social avoidance induced by repeated social defeat stress was significantly attenuated in TRPM2-deficient mice, suggesting that TRPM2 deficiency confers stress resiliency. Our findings indicate that TRPM2 plays an essential role in maintaining behavioral flexibility but it increases susceptibility to stress.

Manipulation of dorsal raphe serotonergic neurons modulates active coping to inescapable stress and anxiety-related behaviors in mice and rats.

Major depression and anxiety disorders are a social and economic burden worldwide. Serotonergic signaling has been implicated in the pathophysiology of these disorders and thus has been a crucial target for pharmacotherapy. However, the precise mechanisms underlying these disorders are still unclear. Here, we used species-optimized lentiviral vectors that were capable of efficient and specific transduction of serotonergic neurons in mice and rats for elucidation of serotonergic roles in anxiety-like behaviors and active coping behavior in both species. Immunohistochemical analyses revealed that lentiviral vectors with an upstream sequence of tryptophan hydroxylase 2 gene efficiently transduced serotonergic neurons with a specificity of approximately 95% in both mice and rats. Electrophysiological recordings showed that these lentiviral vectors induced sufficient expression of optogenetic tools for precise control of serotonergic neurons. Using these vectors, we demonstrate that acute activation of serotonergic neurons in the dorsal raphe nucleus increases active coping with inescapable stress in rats and mice in a time-locked manner, and that acute inhibition of these neurons increases anxiety-like behaviors specifically in rats. These findings further our understanding of the pathophysiological role of dorsal raphe serotonergic neurons in different species and the role of these neurons as therapeutic targets in major depression and anxiety disorders.

Ketamine-Induced Prefrontal Serotonin Release Is Mediated by Cholinergic Neurons in the Pedunculopontine Tegmental Nucleus.

Background: Ketamine rapidly elicits antidepressive effects in humans and mice in which serotonergic activity is involved. Although α4ß2 nicotinic acetylcholine receptor (α4ß2 nAChR) in the dorsal raphe nucleus plays a key role in the ketamine-induced prefrontal serotonin release, the source of cholinergic afferents, and its role is unclear. Methods: Prefrontal serotonin levels after ketamine injection were measured by microdialysis in rats. Electrolytic lesion of pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus was made with constant direct current. Results: Bilateral lesion of the pedunculopontine tegmental nucleus, but not laterodorsal tegmental nucleus, attenuated prefrontal serotonin release induced by systemic ketamine. Intra-pedunculopontine tegmental nucleus, but not intra-laterodorsal tegmental nucleus ketamine perfusion, increased prefrontal serotonin release. This increase was attenuated by intra-dorsal raphe nucleus injection of dihydro-ß-erythroidine, an α4ß2 nAChR antagonist, or NBQX, an AMPA receptor antagonist. Conclusions: These results suggest the ketamine-induced serotonin release in medial prefrontal cortex is mediated by cholinergic neurons projecting from pedunculopontine tegmental nucleus to dorsal raphe nucleus via α4ß2 nAChRs.